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Single‐Cell RNA‐Sequencing Reveals the Breadth of Osteoblast Heterogeneity

The current paradigm of osteoblast fate is that the majority undergo apoptosis, while some further differentiate into osteocytes and others flatten and cover bone surfaces as bone lining cells. Osteoblasts have been described to exhibit heterogeneous expression of a variety of osteoblast markers at...

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Autores principales: Yoshioka, Hirotaka, Okita, Saki, Nakano, Masashi, Minamizaki, Tomoko, Nubukiyo, Asako, Sotomaru, Yusuke, Bonnelye, Edith, Kozai, Katsuyuki, Tanimoto, Kotaro, Aubin, Jane E, Yoshiko, Yuji
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley & Sons, Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8216137/
https://www.ncbi.nlm.nih.gov/pubmed/34189385
http://dx.doi.org/10.1002/jbm4.10496
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author Yoshioka, Hirotaka
Okita, Saki
Nakano, Masashi
Minamizaki, Tomoko
Nubukiyo, Asako
Sotomaru, Yusuke
Bonnelye, Edith
Kozai, Katsuyuki
Tanimoto, Kotaro
Aubin, Jane E
Yoshiko, Yuji
author_facet Yoshioka, Hirotaka
Okita, Saki
Nakano, Masashi
Minamizaki, Tomoko
Nubukiyo, Asako
Sotomaru, Yusuke
Bonnelye, Edith
Kozai, Katsuyuki
Tanimoto, Kotaro
Aubin, Jane E
Yoshiko, Yuji
author_sort Yoshioka, Hirotaka
collection PubMed
description The current paradigm of osteoblast fate is that the majority undergo apoptosis, while some further differentiate into osteocytes and others flatten and cover bone surfaces as bone lining cells. Osteoblasts have been described to exhibit heterogeneous expression of a variety of osteoblast markers at both transcriptional and protein levels. To explore further this heterogeneity and its biological significance, Venus‐positive (Venus(+)) cells expressing the fluorescent protein Venus under the control of the 2.3‐kb Col1a1 promoter were isolated from newborn mouse calvariae and subjected to single‐cell RNA sequencing. Functional annotation of the genes expressed in 272 Venus(+) single cells indicated that Venus(+) cells are osteoblasts that can be categorized into four clusters. Of these, three clusters (clusters 1 to 3) exhibited similarities in their expression of osteoblast markers, while one (cluster 4) was distinctly different. We identified a total of 1920 cluster‐specific genes and pseudotime ordering analyses based on established concepts and known markers showed that clusters 1 to 3 captured osteoblasts at different maturational stages. Analysis of gene co‐expression networks showed that genes involved in protein synthesis and protein trafficking between endoplasmic reticulum (ER) and Golgi are active in these clusters. However, the cells in these clusters were also defined by extensive heterogeneity of gene expression, independently of maturational stage. Cells of cluster 4 expressed Cd34 and Cxcl12 with relatively lower levels of osteoblast markers, suggesting that this cell type differs from actively bone‐forming osteoblasts and retain or reacquire progenitor properties. Based on expression and machine learning analyses of the transcriptomes of individual osteoblasts, we also identified genes that may be useful as new markers of osteoblast maturational stages. Taken together, our data show much more extensive heterogeneity of osteoblasts than previously documented, with gene profiles supporting diversity of osteoblast functional activities and developmental fates. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
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spelling pubmed-82161372021-06-28 Single‐Cell RNA‐Sequencing Reveals the Breadth of Osteoblast Heterogeneity Yoshioka, Hirotaka Okita, Saki Nakano, Masashi Minamizaki, Tomoko Nubukiyo, Asako Sotomaru, Yusuke Bonnelye, Edith Kozai, Katsuyuki Tanimoto, Kotaro Aubin, Jane E Yoshiko, Yuji JBMR Plus Original Articles The current paradigm of osteoblast fate is that the majority undergo apoptosis, while some further differentiate into osteocytes and others flatten and cover bone surfaces as bone lining cells. Osteoblasts have been described to exhibit heterogeneous expression of a variety of osteoblast markers at both transcriptional and protein levels. To explore further this heterogeneity and its biological significance, Venus‐positive (Venus(+)) cells expressing the fluorescent protein Venus under the control of the 2.3‐kb Col1a1 promoter were isolated from newborn mouse calvariae and subjected to single‐cell RNA sequencing. Functional annotation of the genes expressed in 272 Venus(+) single cells indicated that Venus(+) cells are osteoblasts that can be categorized into four clusters. Of these, three clusters (clusters 1 to 3) exhibited similarities in their expression of osteoblast markers, while one (cluster 4) was distinctly different. We identified a total of 1920 cluster‐specific genes and pseudotime ordering analyses based on established concepts and known markers showed that clusters 1 to 3 captured osteoblasts at different maturational stages. Analysis of gene co‐expression networks showed that genes involved in protein synthesis and protein trafficking between endoplasmic reticulum (ER) and Golgi are active in these clusters. However, the cells in these clusters were also defined by extensive heterogeneity of gene expression, independently of maturational stage. Cells of cluster 4 expressed Cd34 and Cxcl12 with relatively lower levels of osteoblast markers, suggesting that this cell type differs from actively bone‐forming osteoblasts and retain or reacquire progenitor properties. Based on expression and machine learning analyses of the transcriptomes of individual osteoblasts, we also identified genes that may be useful as new markers of osteoblast maturational stages. Taken together, our data show much more extensive heterogeneity of osteoblasts than previously documented, with gene profiles supporting diversity of osteoblast functional activities and developmental fates. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. John Wiley & Sons, Inc. 2021-05-17 /pmc/articles/PMC8216137/ /pubmed/34189385 http://dx.doi.org/10.1002/jbm4.10496 Text en © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. https://creativecommons.org/licenses/by/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Articles
Yoshioka, Hirotaka
Okita, Saki
Nakano, Masashi
Minamizaki, Tomoko
Nubukiyo, Asako
Sotomaru, Yusuke
Bonnelye, Edith
Kozai, Katsuyuki
Tanimoto, Kotaro
Aubin, Jane E
Yoshiko, Yuji
Single‐Cell RNA‐Sequencing Reveals the Breadth of Osteoblast Heterogeneity
title Single‐Cell RNA‐Sequencing Reveals the Breadth of Osteoblast Heterogeneity
title_full Single‐Cell RNA‐Sequencing Reveals the Breadth of Osteoblast Heterogeneity
title_fullStr Single‐Cell RNA‐Sequencing Reveals the Breadth of Osteoblast Heterogeneity
title_full_unstemmed Single‐Cell RNA‐Sequencing Reveals the Breadth of Osteoblast Heterogeneity
title_short Single‐Cell RNA‐Sequencing Reveals the Breadth of Osteoblast Heterogeneity
title_sort single‐cell rna‐sequencing reveals the breadth of osteoblast heterogeneity
topic Original Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8216137/
https://www.ncbi.nlm.nih.gov/pubmed/34189385
http://dx.doi.org/10.1002/jbm4.10496
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